Abstract

The genome of the influenza A virus is an eight-segmented negative-strand RNA (vRNA). Progeny vRNAs replicated in the nucleus selectively assemble into a single set of eight different segments, probably in the cytoplasm, and are packaged into progeny virions at the cell membrane. In these processes, a region of approximately 100 nucleotides at both ends of each segment is thought to function as a selective assembly/packaging signal; however, the details of the mechanism, such as the required sequences, are still unknown. In this study, we focused on the 5′-terminus of the sixth neuraminidase gene segment vRNA (Seg.6) to identify the essential sequence for selective packaging. The 5′-terminal region of the A/Puerto Rico/8/34 strain Seg.6 was divided into seven regions of 15 nucleotides each from A to G, and mutations were introduced into each region by complementary base substitutions or synonymous codon substitutions. Mutant viruses were generated and compared for infectious titers, and the relative ratios of the eight segments packaged into virions were measured. We also ascertained whether mutant vRNA was eliminated by competitive packaging with wild-type vRNA. Mutations in the A–C regions reduced infectious titers and eliminated mutant vRNAs by competition with wild-type vRNA. Even under non-competitive conditions, the packaging efficiency of the A or B region mutant Seg.6 was reduced. Next, we designed an artificial vRNA with a 50-nucleotide duplication at the 5′-terminal region. Using this, a virus library was created by randomly replacing each region, which became an untranslated region (UTR), with complementary bases. After selecting proliferative viruses from the library, nine wild-type nucleotides in the A and B regions were identified as essential bases, and we found that these bases were highly conserved in Seg.6 vRNAs encoding the N1 subtype neuraminidase. From these results, we conclude that the identified bases function as the 5′-terminal packaging signal for the N1 subtype Seg.6 vRNA.

Highlights

  • Type A influenza viruses belong to the Orthomyxoviridae family

  • We focused on the 5 -terminal region of Seg.6 virus is an eight-segmented negative-strand RNA (vRNA) (6v5) and aimed to identify the packaging signal sequence required for selective segment assembly and/or packaging

  • It is likely that inter-segmental base pairings and/or RNA secondary structures of these terminal regions are required for selective segment assembly (Shafiuddin and Boon, 2019)

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Summary

Introduction

Type A influenza viruses belong to the Orthomyxoviridae family They have an eight-segmented single-stranded RNA (vRNA) that is encapsulated by a lipid bilayer envelope (Webster et al, 1992; Lamb and Krug, 2001). The 13 and 12 nucleotides (nt) of the 5 - and 3 -terminals of each vRNA, respectively, are common sequences among the eight segments and are thought to form a semi-complementary double-stranded structure. These common sequences bind viral RNA-dependent RNA polymerase (RdRp) comprising PB2, PB1, and PA subunits and serve as the origin for vRNA replication and the promoter for mRNA synthesis. VRNPs together with the viral membrane and matrix proteins form progeny virions

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